Loss of function of the three proteins, KRIT1 (Krev/Rap1 Interacting Trapped 1; CCM1, cerebral cavernous malformation 1), CCM2 (cerebral cavernous malformation 2; OSM, osmosensing scaffold for MEKK3) and CCM3 (cerebral cavernous malformation 2; PDCD10, programmed cell death 10), cause the familial form of the devastating Cerebral Cavernous Malformations (CCM) disease. Loss of function of these proteins is therefore directly linked with stroke, focal neurological defects, seizures and vascula abnormalities. The goal of this application is to understand the molecular underpinnings for normal function of these proteins. To do this we will conduct cell-based, biochemical and structural studies that will address our two central hypotheses: Molecular-level organization of the CCM complex regulates key signaling events and Intra- or inter- molecular head-tail KRIT1 interactions regulate KRIT1 function. In our preliminary studies we have determined the first crystal structures of each of the CCM proteins, KRIT1, CCM2 and CCM3, and have found each of these proteins to contain previously unpredicted protein interaction scaffold domains. Furthermore, our functional studies of the CCM proteins have highlighted important new aspects of their cellular function, particularly with regards to the regulation of integrin activation stat and signaling. Therefore, in Aim 1 we will use our advantaged position to assemble the CCM complex crystallographically and to investigate its functional roles in cells. Our previous studies also investigated the direct interactions of CCM proteins with partners, including ICAP1 and Rap1. These proteins bind KRIT1 and may impact its conformational status, which in turn is suggested to impact formation of the CCM complex. Therefore, in Aim 2 we will discover the molecular mechanisms that regulate KRIT1 conformation and the impact of KRIT1 conformational state on signaling via the CCM complex. In this Multi-Investigator proposal, the Boggon and Calderwood laboratories will conduct a highly collaborative structure-directed functional study of these proteins to better understand their normal functions, with particular attention to CCM disease-related cellular functions. Furthermore, as the CCM proteins are each widely expressed and have high sequence conservation through evolution, we expect that the improved understanding of the CCM proteins obtained from this study will also highlight further roles for the CCM proteins outside of the neurovasculature.